Growth factor receptor bound protein 2 (Grb2) is an adaptor protein that is known to participate in a variety of signal transduction pathways including the MAPK pathway and T-cell activation. We have recently obtained data (J.B.C. & B.B.R.C (in press)) which demonstrate that Grb3-3, an isoform of Grb2 is inducibly and markedly upregulated in CD4+ PBMCs derived from either in vitro HIV-1-infected cultures or HIV-1 -infected human subjects. Analysis of HIV-1 gene products indicates that Tat and Nef can independently induce expression of Grb3L3. We find that Grb3-3 can potentiate NFAT promoter activity in Jurkat T-cells upon engagement of the T-celI receptor (TCR) and CD28 co-receptor. We have also obtained new data which demonstrates that both Tat and Nef can activate NFATc and that Grb3-3 can potentiate HIV-1 LTR activation in the presence of TCR stimulation both of these processes are sensitive to inhibition by cyclosporin A (CyA). In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm and by CyA The goal of this second revised proposal isto define the role(s) of Grb3-3 in HIV-1 replication and in potentiating T-celI activation through NFATc in more detail. Accordingly, the specific aims of this proposal are to use site-directed mutagenesis the critical amino acids of Grb3-3 that are responsible for potentiating the activation of NFATc and to identify a dominant-negative mutant of Grb3-3 that can be used in subsequent blocking experiments of biochemical signaling pathways and of HIV-1 replication. We will use Jurkat T-cells to identify the novel binding partners of Grb3-3, analyze the subcellular distribution of Grb3-3 and determine if its distribution is regulated during T-ceIl activation. We will also confirm that the physiochemical processes identified in Jurkat T-ceII lines are readily translatable to activated, HIV 1-infected CD4+ PBMCs. We will also determine at the biochemical level how Grb3-3 overexpression potentiates NFATc activation through an examination of different potential mechanisms through which Grb3-3 may act including inhibition of MEKK1 activation, inhibition of a MEKK1 activated NFATc kinase which phosphorylates the A domain of NFAT, activation of calcineurin and inhibition of nuclear export of calcineurin (CnA) bound to NFATc perhaps by activation of a nuclear phosphatase or inhibition of a nuclear kinase. We will also determine the critical regions of Tat and Net that are involved in induction of Grb3-3. Finally we will determine if Grb3-3 can modulate HIV-1 replication and reverse transcription through Grb3-3 overexpression studies and inhibition studies using a Grb3-3 dominant-negative mutant and/or an anti-Grb3-3 specific intrabody. These studies will advance our understanding of HIV-1/cellular protein interactions are involved in the pathogenesis of HIV-1-infection and AIDS.